• Vaccine Name: Chimeric SIN/VEE Virus SIN-83
• Target Pathogen: VEE Virus
• Target Disease: Venezuelan equine encephalitis
• Vaccine Ontology ID: VO_0004111
• Type: Recombinant vector vaccine
• Antigen: All structural proteins derived from VEEV TC-83 (Paessler et al., 2003).
• Preparation: The parental pToto1101 plasmid, encoding the SINV genome, and the pTC-83 plasmid, encoding the genome of VEEV TC-83, were obtained from Charles M. Rice (Rockefeller University, New York, N.Y.) and Richard Kinney (Centers for Disease Control, Fort Collins, Colo.), respectively. Fragments containing the SINV subgenomic promoter and the 5′ untranslated region (UTR) of the VEEV subgenomic RNA were generated by PCR amplification, cloned into the pRS2 plasmid for sequencing, and then used for generating the cDNA clone of the chimeric SIN-83S virus genome. The plasmid construct pSIN83 (Paessler et al., 2003) contained the promoter for SP6 RNA polymerase, followed by nucleotides (nt) 1 to 7601 of the SINV genome, nt 7536 to 11382 of VEEV TC-83 (with an additional C→T mutation of nt 7555), an AGGCCTTGGG sequence, and a 355-nt sequence containing the SINV 3′UTR (starting from nt 11394), poly(A) followed by an XhoI restriction site. Plasmids pZPC and pSH, containing infectious cDNAs of VEEV strains ZPC738 (subtype ID) and SH3 (subtype IC), respectively. The plasmids were purified and linearized. RNAs were synthesized and transfected into BHK-21 cells (Bredenbeek et al., 1993). Viruses were harvested after development of cytopathic effects, usually at 24 h following electroporation.
• Virulence: None of the chimeric SIN/VEE viruses caused any detectable disease in adult mice after either intracerebral (i.c.) or subcutaneous (s.c.) inoculation, and all chimeras were more attenuated than the vaccine strain, VEEV TC83, in 6-day-old mice after i.c. infection (Paessler et al., 2003).
• Description: The chimeric SIN/VEE viruses contain the replicative machinery from another alphavirus, Sindbis virus (SINV), and the structural genes from VEEV. The prototype chimeric virus SIN83 is capable of replicating in tissue culture and exhibits a safe and highly attenuated phenotype in mice and hamsters but induces a protective immune response against VEEV (Paessler et al., 2003). It is safe and efficacious in adult mice and hamsters and is potentially useful as VEEV vaccin. In addition, immunized animals provide a useful model for studying the mechanisms of the anti-VEEV neuroinflammatory response, leading to the reduction of viral titers in the CNS and survival of animals.

Mouse Response

• Host Strain: NIH Swiss
• Vaccination Protocol: Six-week-old, female NIH Swiss mice (12 per group) were inoculated on day 0 s.c. into the medial thigh with chimeric SIN/VEE viruses SIN-83. The live VEE TC-83 vaccine virus was used as control for comparison. One half of the animals (six per group) received an additional booster on day 28, which was performed in the same way as the initial immunization. All of the animals were bled on days 1, 2, and 3 and at 4 and 8 weeks after immunization. Serum samples from the first 3 days after immunization were tested for the presence of infectious virus by a plaque assay on BHK-21 cells (Paessler et al., 2003).
• Persistence: To compare the virulence of the VEE TC-83 and SIN-83 viruses, 6-day-old mice were inoculated i.c. or s.c. with different doses of each virus ranging from 2 × 10^4 to 2 × 10^6 PFU. VEEV TC-83 was virulent for weanling mice regardless of the inoculation route. VEEV TC-83 was less pathogenic for weanling mice after s.c. inoculation (mortality rate, 10 to 20%). However, many of the surviving animals developed clinical disease and/or CNS sequelae. None of the SIN-83-inoculated animals had detectable clinical illness. Animals infected with VEEV TC-83 at the age of 6 days were highly inhibited in their growth compared to those infected with SIN-83 or compared to the noninfected control group of the same age (Paessler et al., 2003).
• Immune Response: After 28 days, VEEV-specific neutralizing antibodies in the sera of SIN-83 and VEE TC-83 immunized groups. However, the titers in VEEV TC-83-immunized animals were higher. This can be explained by the higher replication levels of this virus in cell culture.
• Side Effects: none
• Challenge Protocol: Challenge studies to determine the protection against clinical encephalitis in the mouse model. Fifteen 6-week-old, female NIH Swiss mice were vaccinated with 5 x 10^5 PFU of each chimeric virus or PBS alone (control) in a total volume of 100 µl. After vaccination, each cohort of 15 animals was maintained for 8 weeks without any manipulation. Immunized animals were then challenged with VEEV subtype ID strain ZPC738 by using three different inoculation methods: (i) s.c. inoculation into the medial thigh with 10^6 PFU (roughly 10^6 50% lethal dose) per animal in 0.1 ml of PBS (five mice per group), (ii) i.c. inoculation into the left brain hemisphere with 2 x 10^5 PFU per animal in 20 µl of PBS (five mice per group), and (iii) intranasal (i.n.) inoculation with 2 x 10^5 PFU per animal in 20 µl of PBS (five mice per group). Mice were observed for clinical illness (for anorexia and/or paralysis) and/or death twice daily for a period of 2 months.
  
  Challenge studies to determine protection against viral replication in the brain following i.c. or i.n. inoculation with ZPC738. After a group of mice was vaccinated, the first challenge with ZPC738 was performed using two different inoculation methods: (i) i.c. inoculation into the left brain hemisphere with 2 x 10^5 PFU in 20 µl of PBS and (ii) i.n. inoculation with 2 x 10^5 PFU in 20 µl of PBS. Two animals per group were euthanized on days 3, 7, and 28 after infection, and lungs, livers, spleens, kidneys, and brains were collected for viral titration or histological examinations. In addition, 10 animals per group were housed for 28 days after i.n. challenge with ZPC738, without any manipulation. On day 28, all animals from this group received the second i.n. dose of 2 x 10^5 PFU of ZPC738. Two animals per group were euthanized on days 3, 7, and 28 postchallenge, and organs were collected as described above.
• Efficacy: All vaccinated mice were protected against lethal encephalitis following i.c., s.c., or intranasal (i.n.) challenge with the virulent VEEV ZPC738 strain (ZPC738). In spite of the absence of clinical encephalitis in vaccinated mice challenged with ZPC738 via i.n. or i.c. route, high levels of infectious challenge virus in the central nervous system (CNS) were regularly detected. However, infectious virus was undetectable in the brains of all immunized animals at 28 days after challenge (Paessler et al., 2003).

Hamster Response

• Host Strain: golden hamster
• Vaccination Protocol: Three 6-week-old female Syrian golden hamsters per viral strain were vaccinated s.c. in the right medial thigh with 5 x 105 PFU of SAAR/TRD, SIN/ZPC, SIN/TRD, or TC83 strain or PBS alone. Blood samples were obtained daily for the first 3 days after infection, and the animals were observed twice daily for 21 days. Serum viremia was determined by using a plaque assay on BHK-21 cells as previously described. The presence of neutralizing antibody in hamster serum samples was determined via a plaque reduction neutralization test, as described for the murine experiments
• Side Effects: none
• Challenge Protocol: Three weeks after vaccination, the hamsters were challenged s.c. in the medial thigh with ZPC738 at a dose of 106 PFU in a total volume of 100 µl of PBS (roughly 5 x 106 50% lethal dose). The animals were observed for 28 days, and deaths or cases of clinical illness were documented.
• Efficacy: Hamsters vaccinated with chimeric SIN/VEE viruses were also protected against s.c. challenge with ZPC738.
• Description: Six- to 8-week-old female Syrian golden hamsters (Mesocricetus auratus) were purchased from Harlan and acclimatized in the facility for a week prior to infection.